Method of marking a liquid

ABSTRACT

A method of marking a liquid and subsequently detecting that the liquid has been marked, which method comprises: adding to the liquid an additive comprising a plurality of particles in an amount no greater than 1 part weight of particles per 10 6  parts weight liquid, the particles comprising signal means to aid their detection and not being visible in the liquid to the naked eye; sampling a portion of the liquid containing said additive, and detecting the presence of particles in the liquid, with the proviso that said signal means does not consist solely of a nucleic acid tag.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application corresponds to PCT/GB93/01822 filed on Aug. 26, 1993.The latter application is a continuation application claiming priority,in turn, from GB Serial Number 9218131.2 filed on Aug. 26, 1992.

BACKGROUND OF THE INVENTION

This invention relates to the marking of materials and in particular toa method of marking a liquid and subsequently detecting that the liquidhas been marked.

There is a widespread requirement to be able to trace the path taken bya given material as it moves from one location to another. In generalterms, two broad categories of material movement are recognised:

(i) The movement of materials as a result of natural processes occurringin the biosphere, e.g., the flow of water in sub-surface aquifers, themovement of sediments etc.

(ii) The movement of materials which have been manufactured by man,i.e., items which do not occur in the natural environment or which arenatural materials being transported as a result of man's activities. Theformer would include any item produced by man, and the latter items suchas grain and other food materials, mineral ores and petroleum products,such as crude oil.

In all these situations, there may be reasons why it is necessary todevelop specific procedures to trace these movements. It may be thatdirect observation is not possible, e.g., when following the path of anunderground stream. It may be that it is necessary to monitor themovement of goods without the direct knowledge of the transporters or,for legal reasons, to prove that the appearance of a material at aparticular point in the biosphere was due to the same materialoriginating from a known starting point.

For example, it is undesirable and in certain circumstances illegal, forpetroleum materials to leak from storage sites or transportationcontainers and contaminate the natural environment. Petrol storagetanks, e.g., at petrol filling stations, are usually locatedunderground. Should one of these tanks develop a leak, the loss ofmaterial will eventually be detected, either by audits on the materialbeing added to and removed from the storage tank, or by detection ofspilt, leakage material at some site adjacent the storage tank area.Since the tanks are underground, visual inspection is not normallypossible and it is a costly procedure to excavate successive tanks todetermine which tank is the cause of the leakage. The normal procedurewould be to develop a protocol whereby a known marker, e.g., a dye, isadded to the tanks to determine, by tracing the movement of the dye,which tank is the cause of the leakage. Cheaper remedial action can thenbe taken to deal with the identified leaking tank. One feature of thisprocedure is that, in order to know which tank is leaking, the markersadded to each tank must be different, i.e., if there are six tanks, thensix different dyes, each recognisable by some property which can beaccurately and uniquely determined, need to be used. The greater thenumber of individual components in a particular system, the greater thenumber of unique traces that need to be used to make the necessarydistinction between the paths taken by different leaks from differenttanks.

Another example concerns the identification of the source of pollutionin the sea and waterways from spills of petroleum materials,particularly oil. The environmental damage caused by accidental oilspills and deliberate dumping of oil by ships, e.g., when washing tanks,is significant and there is a growing demand for the culprits to beidentified and to be held responsible for clean-up operations. One ofthe problems associated with the identification of oil samples in largevolumes of aqueous media, such as an oil slick on the sea, is that anymarker introduced into oil has a tendency to partition out or bedispersed in the aqueous phase, rendering collection and identificationof the marker particularly difficult.

A further example illustrating the need to monitor the movement of aliquid from one location to another is provided by the practice ofadding to fuel oils, additives, such as antistatic agents, detergentsetc., in order to improve the performance of the oil. It is importantthat persons dealing in such materials are aware whether they have beentreated, but many of these additives are only added in amounts whichcannot be detected without recourse to complex and often expensiveanalytical procedures, and in certain instances it is not readilypossible to determine whether the additive has been added at all,because its presence is effectively masked by impurities in the fueloil. For example, antistatic agents often incorporate chromium ionswhose detection is relatively straightforward. However, naturallyoccurring levels of chromium in oil are often far in excess of thatintroduced by the antistatic agent. It has been proposed to dye the oilto indicate the presence of these additives, but the amount of dye whichmust be added to produce a visible colour change in such materials isunacceptable to both producer and consumer alike, e.g., for reasons ofcost, possible loss of performance, potential damage to engines etc andthe amount may exceed threshold limits set by standards.

Another example is provided by the exemption from value-added-tax (VAT)of fuel oils for agricultural machinery and seagoing vessels. It hasbeen known for unscrupulous individuals to take advantage of thisexemption, by using such fuel for purposes for which there is noexemption, such as motor cars, thereby depriving the government ofrevenue.

In addition, there are many reasons why individuals, corporations,public bodies and governments might wish to mark materials, e.g., tomonitor the flow of materials along distribution and sales networks, inorder to be able to determine the ultimate fate of that material and/orthe efficiency of a particular distribution network compared withanother.

Many tracing methods have been used to solve problems of this sort, allof which involve the addition of some characteristic marker, such asdyes or radioactive compounds, to the material being monitored.Biological materials, such as bacteriophage or bacteria have also beenused, most notably for tracing the movement of water bodies in thenatural environment. In these cases, the living systems possess someproperty (e.g., a known drug resistance pattern or particular hostspecificity) which does not normally occur in nature. The addedorganisms can be traced from their point of addition by obtainingsamples as required, isolating any organisms in those samples, andshowing that the organisms originally added can be isolated from thesamples.

International Patent Publication No. WO 87/06383 discloses a method oflabelling an item or substance which involves labelling with amacromolecule, such as nucleic acid or a polypeptide. The method takesadvantage of the ability to detect the presence or absence of molecules,such as DNA or protein per se, by simple chemical analytical procedures,referred to as "YES/NO" tests, which indicate whether or not themacromolecule is present. For example, the presence of DNA can bedetected by using non-specific chemical agents which bind to the DNA,such as ethidium bromide, acridine orange or bis-benzimide (H33258,Heochst dye 33258). In the case of ethidium bromide, this compoundcannot be detected under normal visual light wavelengths. Labelling maytherefore be achieved by providing DNA and ethidium bromide together.The presence of the DNA (with bound ethidium bromide) can subsequentlybe detected by ultraviolet irradiation. There is no discriminationbetween different DNA molecules from different sources, e.g., fromdifferent organisms.

The resolution of the system may, however, be considerably improved bytaking advantage of the ability of macromolecules, such as nucleic acidsand proteins, to be recognised unequivocally by a second complementarymacromolecule to provide a unique marker. Accordingly, it is possible todetermine the authenticity of an item or substance, by labelling thatitem or substance with a predetermined macromolecular first compoundcapable of binding to a second complementary macromolecular compound andusing that second compound as a probe to determine the presence orabsence of the first compound and thus establish whether a given item orsubstance is the genuine (marked) article.

The uniqueness of DNA to each species and, indeed, each strain within aspecies, together with the technical capacity to hybridise unique DNAmolecules provides a more sophisticated form of labelling than a simple"YES/NO" test. For each strain of organism, the DNA (or RNA) moleculesare unique, although different strains of the same species differ byvirtue of small variations in sequences of bases. It is possible torecognise the DNA of different species and different strains of the samespecies by examining the DNA with labelled DNA probes. An item orsubstance may be labelled with a "signal DNA" comprising a sequencecapable of hybridising with a specific "probe DNA". Both the signal DNAand the probe DNA are kept secret. Where analysis of the labelled itemor substance by means of the probe DNA reveals the signal DNA, the itemor substance is genuine. If not, the item or substance is an imitation.

This marking technique is primarily intended for labelling articles,such as luxury goods, e.g., watches, perfume and clothes; films andrecordings; bank notes; art works; documents such as passports, andmachinery and parts, e.g., for cars, although reference is made tolabelling pharmaceuticals and other chemicals, such as fertilisers,herbicides and pesticides.

Labelling may be achieved in a variety of ways, e.g., the signalcompound may be incorporated directly into the item or substance duringits manufacture, or it may be attached by an adhesive. The signalcompound may also be included in a material such as a paint or ink whichis applied to an item or substance.

International Patent Publication No. WO 90/14441 discloses a method ofmonitoring the presence of a substance which comprises marking thesubstance with a nucleic acid tag, collecting the substance anddetecting the tag, generally by amplifying the nucleic acid usingpolymerase chain reaction technology. The polymerase chain reaction(PCR) procedure is disclosed in, e.g., U.S. Pat. Nos. 4,683,202 and4,683,195, and European Patent Publication Nos. 258017 and 237362, andallows for the enzymatic amplification, in vitro, of specific DNAsequences using oligonucleotide primers which recognise all or part ofthe DNA molecule used as the taggant. The use of PCR technology enablesthe DNA molecule to be amplified exponentially, e.g., 25 complete cyclesof amplification enables a single DNA molecule to be increased 3.4×10⁷times.

Also disclosed is a kit designed to tag and monitor substancescomprising a nucleic acid taggant and a polynucleotide complementary tothe taggant which can be either a signal probe, capture probe or aprimer for the PCR method. Reference is made to the kits containing"signal means", such as enzymes, radio-isotopes and fluorescent labels,but no further details are provided.

Substances which may be tagged are said to include air pollutants,organic solvents (such as those from dry cleaners, chemical factories,airports and petrol filling stations), explosive compositions (such asplastic explosives and gunpowder), paper goods (such as newsprint, moneyand legal documents), pharmaceutical products (such as medicaments),inks, perfumes and paint products.

The nucleic acid may be free, i.e., naked, encapsulated by polymericsubstances (such as proteins) or lipophilic compositions (such asliposomes), bound to a component of the tagged substance or bound to asolid support which is then mixed with the substance being tagged.Suitable support materials are said to include latex, dextran andmagnetic beads, but no further details are provided.

Our copending International Patent Publication No. WO91/7265 alsodiscloses a method for tracing the origin and movement of materials,both liquid and solid, which comprises: adding to the material amicrotrace additive comprising DNA molecules; sampling the resultingmaterial after movement thereof, and detecting the presence of themicrotrace additive in the sample.

In a preferred aspect of the invention, the material being monitored isa liquid hydrocarbon, such as oil, and the microtrace additive isdesigned such that it cannot be easily removed from the hydrocarbon byaqueous washing, e.g., following an oil spill at sea. In mixtures ofwater and hydrocarbons, any DNA present in the hydrocarbon tends to moveto the aqueous phase. The partitioning of DNA under these conditions isdue to the negative charges associated with the phosphodiester groups ofthe DNA and the ability to form hydrogen bonds with water molecules andan inability to do so in a hydrocarbon environment. Various methods areproposed for ensuring that the DNA remains in the hydrocarbon ratherthan partitioning to any aqueous phase, including covalently linking theDNA to hydrophobic beads, typically of from 1 to 5 μm diameter, designedto be soluble in hydrocarbons and not the aqueous phase.

By taking advantage of recent advances in techniques, such as PCRtechnology, for the detection of DNA at exceedingly low concentrations,only small quantities of DNA, typically in the concentration range1×10⁻¹¹ to 1×10⁻⁶ g DNA per ml of oil or other liquid, are used in themicrotrace additive. For example, plasmid pBR322 DNA (2×10⁻⁹ g), chosenbecause DNA primers for amplification of this molecule are commerciallyavailable, was added to Arabian light crude oil (100 μl) and mixed. Tosubsequently extract the DNA, distilled water (100 μl) was added to theoil and the mixture thoroughly mixed to extract the pBR322 DNA from theoil into the aqueous phase. The oil-water mixture was centrifuged (10000xg for 5 minutes) and the aqueous phase layer (5 μl) removed and loadedinto a standard Tag polymerase PCR reaction vial and reaction mixture(100 μl containing KCl (50 mM); Tris-HCl buffer (10 mM; pH8.4); MgCl₂(1.5 mM); gelatin (100 μg/ml); two pBR322 DNA primers (0.25 μm);deoxyribose nucleotide phosphates (200 μg of each of dATP, dCTP, dGTP,dTTP), and Tag polymerase (2.5 units). Following automated PCR cycling,the reaction mixture (10 μl) was loaded onto agarose gel (2% w/v) andelectrophoresed under standard conditions. The completed gel was stainedwith ethidium bromide to visualise the amplified DNA. No bands appearedin various negative controls.

Whilst DNA is particularly suitable for use as a unique marker, thereare many instances where all that is required is a simple "YES/NO" testof the type described previously, e.g., to indicate that a particularfuel oil has been treated with a certain additive etc. In suchcircumstances, DNA is a less effective marker, as the DNA must either bepresent in prohibitively large amounts for it to be detected bynon-specific assays, such as ethidium bromide staining, or PCRtechniques are required to increase the amount of DNA to a level whichcan be detected. Thus, there is a continuing need for an accurate,reliable and cost-effective method of marking a liquid which is capableof providing a "YES/NO" test, and which does not rely on the use ofcomplex, time-consuming analytical procedures or the use of unacceptablyhigh levels of marker.

Many of the immunodiagnostic assays performed in clinical laboratoriesutilise a bioreactive molecule, typically an antibody, having a specificbinding affinity for a target molecule, e.g., the antigen in respect ofwhich the antibody was raised, in order to identify and/or isolate thattarget molecule in a given test sample. The bioreactive molecule isoften coupled to the surface of a microbead, in order to increase thetotal surface area available to capture the target molecule and tofacilitate the separation of bound target molecules from a solution offree molecules, since they can easily be immobilized, e.g., on a filter.Such beads are typically formed of a polymeric material, and generallyhave a diameter within the range from 0.05 to 100 μm. The beads may beprovided with a label, such as a fluorescent label, radiolabel etc., toprovide signal means. Other beads are magnetic to aid their separationfrom the test sample, e.g., a magnet can be used to pull the beads intoone region of the test vessel from which they can be physicallyseparated. Magnetic beads can be prepared by dispersing particles of amagnetic material, such as magnetite (Fe₃ O₄), into the polymericmaterial used to form the particles.

Such microbeads are widely used in several fields of biochemistry andmedicine, including the isolation of cells and target molecules fromwhole blood, tissue extracts, tissue cultures, enzyme digests and solidtissues; tissue typing; the isolation of PCR or Klenow DNA fragments; ascarriers for pharmaceutical preparations; the separation of cancer cellsfrom healthy cells; to provide a ready prepared template for genomewalking, and the selective enrichment and/or isolation of pure andviable micro-organisms or smaller target compounds like solubleantigens, e.g., as disclosed in British Patent Publication No. 2017125,U.S. Pat. Nos. 4,035,316, 4,105,589, 4,138,383, 4,186,120, 4,224,198,4,259,223, 4,267,237, 4,326,008, 4,369,226, 4,410,370, 4,510,244,4,530,956, 4,550,017, 4,552,812, 4,563,510, 4,622,362, 4,654,267,4,654,300, 4,663,277, 4,678,814, 4,689,307, 4,783,336, 4,828,984,4,962,023, 5,028,545, and 5,081,020, and European Patent PublicationNos. 91453, 10986 and 106873.

Microbeads bearing fluorescent labels are commonly used to align,calibrate and correct apparatus, such as fluorescence microscopes andflow cytometers, e.g., as disclosed in U.S. Pat. Nos. 4,224,359,4,714,682, 4,774,189, 4,857,451, 4,868,126, 4,918,004, 5,073,497,5,084,394 and 5,093,234.

SUMMARY OF THE INVENTION

The present invention seeks to provide an alternative method for themarking of liquids.

According to one aspect of the invention there is provided a method ofmarking a liquid and subsequently detecting that the liquid has beenmarked, which method comprises:

adding to the liquid an additive comprising a plurality of particles inan amount no greater than 1 part by weight of particles per 10⁶ partsweight liquid, the particles comprising signal means to aid theirdetection and not being visible in the liquid to the naked eye;

sampling a portion of the liquid containing said additive, and

detecting the presence of particles in the sample, with the proviso thatsaid signal means does not consist solely of a nucleic acid tag.

In the context of the present invention, any reference to the particles"not being visible in the liquid to the naked eye" is to the individualparticles, when dispersed in the liquid, not being visible withoutrecourse to optical aids, such as microscopes.

The term "liquid" should be construed sufficiently broadly to encompassviscous and semisolid materials, such as tars, bitumen resins, paintproducts, syrups etc. It should also be construed as encompassing liquidmaterials which are subsequently stored, transported or used in solid orsemi-solid form, e.g., inks, paint products etc.

The samples need not be drawn from the main body of the liquid, but fromthe environment, e.g., the sea in the case of an oil spill, nor do thesamples have to be in the form of a liquid, e.g., where a waste materialhas been illegally discharged into the soil, samples of earth may berecovered, even after a period of time has expired, and analyzed.

The term "hydrocarbon" is to be construed broadly as relating to anyorganic compound having as a major component thereof carbon andhydrogen, thereby encompassing not only compounds consisting solely ofcarbon and hydrogen, including both aliphatic and aromatic and saturatedand unsaturated compounds, but also compounds containing heteroatoms,such as oxygen, nitrogen, sulphur, selenium, vanadium etc., e.g.,alcohols, ethers and the like.

The term "oil" should be construed as describing any water-insoluble,liquid, including those derived from petroleum, coal, shale etc., bydistillation, cracking and chemical treatment, and fixed (or fatty) oilsobtained from animals and plants, such as olive oil, palm oil, rapeseedoil, sunflower oil, whale oil etc.

The method of the invention provides an accurate, reliable andcost-effective method of marking a liquid, which may be used as a simple"YES/NO" test or, if desired, as a more specific test for tracing theorigin and/or movement, from one location to another, of liquids. Themethod can be used to mark substantially any liquid, although for mostpurposes it use will be confined to more valuable liquids, such as crudeoil, fuel oils, e.g., petrol, diesel oil, paraffin, aviation fuel etc.In addition to hydrocarbons, the present invention finds utility in themarking of liquids as diverse as perfumes, inks, paint products,pharmaceuticals and other chemicals, such as fertilisers, herbicides,pesticides and organic solvents, waste discharges from factories,refineries, power stations, nuclear waste etc.

According to a further aspect of the invention there is provided aliquid containing an additive comprising a plurality of particles addedin an amount no greater than 1 part by weight of particles per 10⁶ partsweight liquid, the particles comprising signal means to aid theirdetection and not being visible in the liquid to the naked eye, with theproviso that where said signal means comprises a nucleic acid tag,either the particles further comprise a second different signal means orsaid additive also comprises particles having signal means comprisingother than a nucleic acid tag.

DETAILED DESCRIPTION OF THE INVENTION

The choice of particles for use as the additive is primarily dependenton the type of liquid being marked. For example, differentconsiderations arise when marking crude oil when compared with perfume,both in terms of the nature of each material, e.g., viscosity (specificgravity), hydrophobicity, opacity etc., the manner in which the materialis treated, stored and transported and the purpose to which the materialwill be put. Obviously, there are far less restrictions on what can beadded to a shipment of crude oil for refining than to a perfume, and thelogistics of marking a 250,000 tonne shipment of crude oil are verydifferent from those for marking 250 ml bottles of perfume. For example,in the former case, it is important that the particles are evenlydispersed throughout the entire cargo if it is to be used to trace theguilty party in the event of an oil spillage, whereas in the lattercase, the bottle of perfume need only be shaken prior to sampling whentesting, e.g., the wares of a street trader suspected of peddling stolengoods.

The density of the particles is advantageously matched with the specificgravity of the liquid being marked to ensure that the additive will,once added to the liquid, remain evenly distributed throughout theliquid. However, in relation to oil carried by tankers, settling will becounteracted to some extend by pumping and slopping of the cargo. Evendistribution is an important consideration where, e.g., it is intendedto discourage illegal activities, such as black marketeering, thewashing of oil tanks at sea etc., or where the liquid is subsequently tobe divided into smaller volumes.

The particles should be compatible with the liquid, e.g., when markingoils and other hydrophobic materials, the particles should be ofhydrophobic (lipophilic) character to minimise the possibility of theirpartitioning into the sea in the event of a spillage. The particlesadvantageously do not dissolve in the liquid, but form a very finedispersion to allow their subsequent separation from the liquid.

The particles may have any size or shape appropriate for the intendedpurpose, e.g., they may be solid or hollow, of regular or irregularshape etc., although for most purposes they preferably constitute ahomogeneous population of substantially identical size, shape, densityetc., such that the behaviour of the particles in the liquid can bepredicted.

The particles are added to the liquid in an amount no greater than 1part by weight particles per 10⁶ parts weight liquid, although it willbe appreciated that it is sometimes necessary to add the particles ingreater amounts, e.g., in the case of a concentrate, in anticipation ofsubsequent dilution. The particles are preferably added in an amount nogreater than 1 part by weight per 10⁸ parts weight liquid, morepreferably in an amount no greater than 1 part by weight particles per10¹⁰ parts weight liquid. The particles are typically added to theliquid in an amount of from about 1 part by weight particles per 10¹⁰parts weight liquid to about 1 part by weight particles per 10¹² partsby weight liquid.

The particles may be formed of any suitable non-living or non-viableformerly living matter material, including (but not limited to):polymeric materials (whether synthetic or naturally occurring), ceramicmaterials, glasses and the like, with the general proviso that theparticles are inert, i.e., non-reactive, to the liquid being marked.Polymeric materials are preferred and examples of suitable polymericmaterials include (but are not limited to): polyether sulphones;polyimides, such as polyimide-amides and polyether imides;polysulphones; cellulose esters, such as ethyl cellulose, celluloseacetate, celulose acetate hydrogen phthalate, cellulose acetatebutyrate, cellulose acetate propionate, cellulose triacetate etc.;polyvinyl resins, such as poly(vinyl acetate), poly(vinyl chloride),poly(vinyl pyridine), poly(vinyl alcohol) etc.; polyacetals, such aspoly(vinyl butyral), poly(vinyl formal) etc.; polyesters, such aspoly(ethylene terephthalate), poly(ethylene naphthalate) etc.;fluorinated polymers, such as poly(vinylidene fluoride),poly(tetrafluoroethylene), poly(tetrafluoroethylene-hexafluoropropylene)etc.; polyacrylates, such as polyacrylic acid, polymethacrylic acid,polymethylmethacrylic acid etc; latex and other rubbers or gums;polycarbonates; polyolefins, such as polyethylene, polypropylene,polystyrene etc; polyamides, such as nylon, and dextran, starch andother polysaccharides.

In a preferred embodiment of the invention, the particles comprisemicrobeads or microspheres. Exemplary microbeads/spheres arecommercially available from Dynal (U.K.) Ltd. of Wirral, Merseyside,U.K., under the generic tradenames DYNABEADS and DYNASPHERES. Thepreparation of these beads is disclosed in, e.g., European PatentPublication Nos. 91453, 10986 and 106873 and U.S. Pat. Nos. 4,186,120,4,530,956, 4,563,510 and 4,654,267.

The particles are preferably present such that there are on average notmore than 1000 particles per ml of liquid, more preferably not more than100 particles per ml of liquid and most preferably between 1 and 100particles (inclusive) per ml of liquid, with a typical amount about 10particles per ml.

The particles may have any size suitable for the intended purpose, withthe proviso that individual particles should not be visible (in theliquid) to the naked eye. Generally, particles having an average sizenot greater than about 5 μm are suitable for most purposes. Theparticles preferably have an average size of from 0.01 to 5 μm, morepreferably 0.05 to 1 μm, with a typical size about 0.25 or 0.5 μm.

The particles may advantageously be of such a size that they exhibitBrownian motion in the liquid. This phenomenon may be used to aid theformation of a substantially uniform distribution of particlesthroughout the liquid.

The signal means to aid the detection of the particles in the liquid maytake a wide variety of forms, but is preferably of the type that willallow the person testing the liquid to determine the presence or absenceof the particles relatively quickly, preferably within a few minutes andcertainly within a few hours. The detection procedure preferably doesnot involve the use of complex analytical procedures and techniques,although some experimental manipulation is inevitable. The testingprocedure is preferably such that it can be conducted on site, i.e., onboard a marine tanker, at the site of a storage tank etc., withoutsending samples to a laboratory.

The following recitation is provided by way of example only and shouldnot be considered to be exhaustive:

(1) The particles may be magnetic. A sample of liquid suspected ofcontaining magnetic particles can be analyzed, e.g., by using a magneticprobe to extract the particles from the liquid. The isolated particlescan then be further analyzed. Alternatively, a magnet can be used topull the beads into one region of the test vessel from which they canphysically separated. Suitable magnetic beads are commercially availablefrom Dynal (U.K.) Ltd. of Wirral, Merseyside, U.K., under the generictrade name DETACHaBEAD, and are disclosed in, e.g., U.S. Pat. No.4,654,267. Apparatus for the separation of magnetic microspheres islikewise available from Dynal (U.K.) Ltd., under the trade names MCP-1,MCP-6 and MCP-E.

(2) The particles may have a known size or shape distribution to allow aparticular batch to be identified, by determining the frequency ofparticles of one size or shape relative to the other. Particle size(volume) can be determined by the Coulter principle based on the changein electrical impedance due to each particle, and can be used todistinguish particles of identical or overlapping size ranges, providedthe particles have different impedance characteristics. Labels providingsignificant differences in electrical impedance, e.g., metal particles,such as gold, may be used to provide such a signal. Thus, particle-basedassays can be performed using a Coulter counter without having toseparate the particles prior to testing.

(3) The particles may be coloured, e.g., by dispersing appropriatepigments into the beads during their preparation, although this isgenerally only practical for larger particles. The additive may compriseparticles of a single colour or a number of colours, with thedistribution of the differently coloured particles selected to allow aparticular batch to be identified, by determining the frequency of thedifferent coloured beads in given sample.

In a preferred aspect of the invention, the particles are used toconcentrate, in the region of the particle, what would otherwise be verylow amounts of a signal label, i.e., amounts which, if uniformlydispersed throughout the liquid, would produce a concentration of labeltoo low to be readily detected. This aspect of the invention will now bedescribed with reference to (4) to (6) below.

(4) The particles may be provided with a fluorescent, luminescent orphosphorescent label. "Fluorescence" describes the emission of light ofa different (usually greater) wavelength by a substance followingexposure to exciting radiation. "Luminescense" describes the emission oflight under the influence of various physical agents, e.g., chemicalagents (chemiluminescence) etc. "Phosphorescence" describes theemission, usually after a defined interval, of light by a substancefollowing exposure to heat, light or electric discharge. It will beappreciated that these terms are not mutually exclusive and there issome overlap between such labels.

The preferred signal means for use in the method of the invention arefluorescent substances, especially fluorescent dyes, e.g., of the typecommonly used in fluorometric flow cytometry. Suitable fluorescent dyesinclude (but are not limited to): allophycocyanine, phycocyanine,phycoerythrine, rhodamine, oxazine, coumarin, fluoroscein derivatives,e.g., fluorescein isothiocyanate and carboxyfluoroscein diacetate, aswell as Texas red, acridine yellow/orange, ethidium bromide, propidiumiodide, bis-benzamide (commerciallly available from Hoechst under thetrade name H33258) etc. A sample of liquid suspected of containingparticles bearing a fluorescent label may be easily and rapidly analyzedusing, e.g., a fluorescence microscope or a flow cytometer.

The additive may contain two (or more) types of particles, each typebearing a differently coloured label. Qualitative differences in thesignals from the labels, e.g., fluorescence wavelength, will distinguishthe respective particle populations. The distribution of the particletypes may be selected such that it is possible, by examining thefrequency of each label in a given sample, to identify a particularbatch of liquid.

Particles capable of emitting light following irradiation by excitingradiation can be amplified using, e.g., a photomultiplier. Thistechnique is especially useful if the light emitted by the particles isof a different wavelength to the exciting radiation, as is the case withphosphorescent labels. A laser may be used as the irradiating source.Alternatively, polarised light may be used.

Conventional flow cytometers use light scattering to detect eachparticle and, as the light scattering signal is proportional to particlesize, particles of different sizes can also be distinguished, providingthe size-ranges of the respective populations do not overlap. Ingeneral, the concentration of particles in an unknown sample can bedetermined by measuring, the fluorescence intensity of the particles andreading the corresponding concentration from a standard curve (whereparticle concentration is a function of fluorescence intensity). Theparticles of each population are preferably uniform in size as well assurface area characteristics, since this results in less variance influorescence per particle. The aforedescribed DYNOBEADS and DYNOSPHERESare perfect spheres with a relative standard deviation (CV) in lightscatter measurements of about 1%. A number of such particle types cantherefore be mixed and still easily identified as non-overlappingpopulations in a flow cytometric light scatter histogram. Thus, readingof particle-based assays can be performed by flow cytometry withouthaving to separate the particles prior to reading.

Other labels providing a photometric signal, including colloidal goldparticles etc., may also be used.

(5) An enzyme may linked to the particles. Suitable enzymes and assayprocedures are well known, but useful examples include (but are notlimited to): alkaline phosphatase or other transferase, catalase,β-galactosidase, horseradish peroxidase and luciferase. A sample ofliquid suspected of containing particles bearing an enzyme can beanalysed by addition of that sample or, if the liquid, e.g., oil, doesnot allow direct addition of the sample, as most enzyme reactions areaqueous based, the isolated particles, to a reaction mixture containingthe appropriate substrate and such enzyme cofactors as are necessary,and monitoring the reaction catalysed by the enzyme, e.g., by theappearance of a reaction product or the removal of the enzyme substrate.

For example, referring to the above exemplified enzymes, luciferase canbe detected by the emission of light caused by the breakdown of ATP toADP+P.

β-galactosidase can be detected spectrophotometrically using "X-gal"[5-bromo-4-chloro-3-indolyl-β-D-galactoside] which is a colourless,chromogenic substrate cleaved by β-galactosidase to release a blueindolyl derivative. The use of β-galactosidase and X-gal is well knownin bacteriology.

Any enzyme, such as alcohol oxidase, aldehyde oxidase, amino-acidoxidase, ascorbate oxidase, galactose oxidase, glycollate oxidase,glucose oxidase, hexose oxidase, lactate oxidase, malate oxidase, NADHoxidase, oxalate oxidase, pyruvate oxidase, tryptophan oxidase, urateoxidase and xanthine oxidase which, directly or indirectly, consumes orrequires oxygen, can be monitored by measuring the rate of oxygen uptakeor evolution. For example, glucose oxidase catalyzes the consumption ofoxygen according to the amount of glucose available, as expressed by theequation:

    C.sub.6 H.sub.12 O.sub.6.H.sub.2 O+O.sub.2 →C.sub.6 H.sub.12 O.sub.7 +H.sub.2 O.sub.2

The resulting decrease in oxygen can be sensed by an oxygen electrode.Redox dyes directly coupled or indirectly coupled through anenzyme-glucose reaction could also be used to provide a colorimetricchange.

The enzyme may produce hydrogen peroxide as a by-product which can besensed by a hydrogen peroxide sensitive electrode, e.g., a H₂ O₂polarographic anode. A colorimetric method may be used for detectingamounts of hydrogen peroxide produced by the enzyme reaction, e.g., theamount of hydrogen peroxide produced may be measured by a system whichcomprises a chromogenic reagent or reagents capable of undergoing acolour change in the presence of hydrogen peroxide. One known method ofsuch measurement is by means of a quadravalent-titanium and xylenolorange which react to form a stable red colour with hydrogen peroxide(Taurenes & Nordschow, American Journal of Clinical Pathology, Vol. 49,p. 613, 1968). The amount of hydrogen peroxide produced is measured bythe intensity of the colour. Alternatively, an enzyme such as catalasewhich reacts with hydrogen peroxide according to the following reactionscheme:

    2H.sub.2 O.sub.2 →2H.sub.2 O+O.sub.2

can be monitored by measuring the amount of oxygen evolved or theremoval of the hydrogen peroxide.

The reaction may also be followed by measuring the electrons which areremoved during the enzyme reaction and transferred to a coloured dye,e.g., lactic acid dehydrogenase removes electrons from lactic acid whichare then available for transfer to a coloured dye. Alternatively,electrons removed during the enzyme reaction may be transferred directlyto an appropriate "biosensor" which generates an electronic signalproportional to enzyme activity. Suitable biosensors are well known inthe field of biochemistry and provide a much simpler way of quantifyingenzyme activity when compared with colorimetric methods.

A pCO₂ electrode may be used to measure the carbon dioxide evolved fromthe action of decarboxylases, such as acetoacetate decarboxylase,arginine decarboxylase, aspartate decarboxylase, glutamatedecarboxylase, lysine decarboxylase and pyruvate decarboxylase.

(6) The signal means may comprise a radiolabel. A sample of a liquidsuspected of containing particles bearing a radiolabel can be analyzedusing a Geiger-Muller tube or scintillation counter, or by coating athin film of the liquid onto an appropriate substrate and overlaying itwith a photographic film, the radiolabel causing fogging of the film inthose regions immediately adjacent the particles. The radiolabel must beadded in amounts greater than the naturally occurring radioactivity ofthe liquid. Suitable radiolabels are well known in the field ofbiochemistry, e.g., ³² P, ³⁵ S and ¹²⁵ I.

The attachment of radiolabels, enzymes and the like to particles, iswell known in the context of immunodiagnostic kits etc., and will not bedescribed herein.

The particles may have to be removed from the liquid prior to anytesting, depending on the nature of the liquid and the type of signalmeans used. This is especially true of enzymatic labels which areusually aqueous based. Separation of the particles may be accomplishedby a wide variety of techniques, e.g., centrifugation, filtration, theuse of a magnet to separate magnetic particles, column chromatographyetc. Alternatively, the particles may be coated with a molecule having astrong binding affinity for another molecule. The particles may beremoved or concentrated by passage through a column comprising thatother molecule bound to a solid support matrix or the sample may bewashed over a substrate, e.g., a microscope slide, to which that othermolecule has been anchored. Suitable pairs of binding molecules include(but are not limited to): antigen and specific antibody; hormone andhormone receptor; hapten and antihapten; polynucleotide andcomplementary nucleotide; polynucleotide and polynucleotide bindingprotein; biotin and either of avidin and streptavidin, especiallystreptavidin; enzyme and enzyme cofactor, and lectin and specificcarbohydrate.

The use of streptavidin and biotin is especially preferred, asstreptavidin has a very high binding constant (almost irreversible).Particles bearing one of avidin/streptavidin and biotin may beconcentrated by a procedure, such as column chromatography, therebyenabling more dilute dispersions of the additive to be used, or simplermethods for the detection of the appropriate label carried by theparticles.

(7) These surface bound molecules can also be used as a means to aiddetection of the particles in their own right. For example, usingtechniques similar to those employed in indirect (or sandwich)immunoassays, a reagent containing one of each pair of specific bindingmolecules bearing a label, e.g., an enzyme, fluorolabel, radiolabeletc., may be added to the sample suspected of containing particles. Anyparticles present are then isolated and washed to remove unbound reagentand the presence of the label detected as described previously.Alternatively, a probe coated with one of a pair of specific bindingmolecules can be used to extract particles coated with the complementarymolecule from the liquid. If necessary, the probe and beads can beexamined under a microscope.

(8) The additive may comprise two (or more) different types ofparticles, each having a different label from that of the other, e.g.,the combination of a fluorescent label and a radiolabel. The number ofparticles of each type present in the sample may be estimated bycomparing the results obtained against standard curves prepared in thelaboratory. Thus, by measuring the frequency of each label in a givensample, it is possible to identify a particular batch.

(9) The particles may be formed of a material having a different thermalconductivity to the liquid being marked, such that they emit differentamounts of heat compared to the surrounding liquid. Such particles canbe visualised using infrared (IR) image analysis techniques. Theadditive may contain two (or more) types of particles having widelydifferent thermal conductivities. Qualitative differences in the heatemitted by the particles will distinguish the respective particlepopulations. The distribution of the particle types may be selected suchthat it is possible, by examining the frequency of each particle in agiven sample, to identify a particular batch of liquid.

(10) Microscopic analysis of a liquid sample suspected of containingparticles can be conducted using any of the well known techniques ofmicroscopy, including light microscopy, phase-contrast microscopy,electron microscopy etc. Phase contrast microscopy, well known from thefield of bacteriology generally provides better visualisation ofnon-labelled particles.

The aforedescribed signal means are primarily intended as a "YES/NO"test, i.e., to indicate the presence of absence of particles in aliquid, although by using a mixed population of particles, it ispossible to introduce a degree of specificity into the protocol.However, to provide a test to indicate the origin of a particularsample, e.g., to allow the authorities to identify the party responsiblefor an oil spillage, it is preferred to provide the particles with aunique marker, typically a macromolecule, such as a nucleic acid orpolypeptide, preferably the former to take advantage of PCR technology.

The second (unique) marker may be present on the same particles as thatof that of the non-specific marker or on different particles which mayor may not have the same size and/or shape of the other particles.

The tagging of substances with nucleic acid is known and disclosed in,e.g., International Patent Publication Nos. WO87/06383 and WO90/14441and our own copending International Patent Publication No. WO91/17265(hereby incorporated by reference). The tagging of substances withpolypeptides and proteins is also known and disclosed in, e.g., U.S.Pat. Nos. 4,359,363 and 4,441,943. In the former case, the nucleotidebase sequence is used to provide a means to encode information, whereasin the latter, it is the sequence of amino acids which to encodes theinformation.

Nucleic acids can provide a limitless amount of information, because ofthe variable sequence of bases (adenine, cytosine, guanine and thymine[uracil in the case of RNA which replaces thymine]) contained within themolecule. Probability terms can be calculated for the frequency of agiven sequence of bases and, so long as sufficient bases are used, i.e.,a sufficiently large DNA molecule is employed as the taggant, then forall practical purposes a unique microtrace can be defined. By usingcombinations of universal sequences (accepted as industrial standards)and by varying levels of specific sequences, it is possible to identitythe type of generic product, the product's origin (company specificsequences), the lot or batch, and even provide an identifier for a unitof commerce.

Both naturally occurring and synthetic nucleic acids are suitable foruse as the taggant. They can be single or double stranded. The term"naturally occurring" refers to DNA (or RNA) molecules occurring innature. An example of naturally occurring DNA molecule is pBR322, forwhich a known sequence has been determined (by DNA sequencingprocedures). The term "synthetic" is applied to DNA (or RNA) synthesizedin the laboratory using routine synthesis procedures well known in therelevant art.

Synthetic DNA may be formed from the five naturally occurring bases:adenine, thymine, guanine, cytosine and uracil, and non-naturallyoccurring bases, e.g., inosine bases, and derivatized nucleotides, suchas 7-deazo-2'deoxyguanosine, alkylphosphonate oligodeoxynucleotides,phosphorothioate oligodeoxynucleotides and α-anomericoligodeoxynucleotides. In certain circumstances, taggants incorporatingnon-naturally occurring bases may have advantages over those containingonly naturally occurring bases, e.g., in stability etc, because they areless likely to be degraded by nuclease activity, by chemically activesubstances or by environmental conditions, such as heat or ultravioletradiation. The use of taggants incorporating non-naturally occurringbases is limited only by their ability to be effectively detected by theselected detection means. For tagging methods using the preferred PCRtechnology, the taggant must be capable of forming duplexes with PCRprimers and function as a template for the polymerases used in the PCRprocedure.

The preferred molecular structure of the nucleic acid taggant will varywith the means used to detect the nucleic acid. Typically at least 20nucleotide bases are necessary to ensure adequate specificity for anytaggant so that accidental contamination will not lead to false results.The longer the sequence, the higher the potential information content ofthe taggant, but the more likely that degradation will become a problem.Typically, fragments under 1 kilobase are preferred.

Because of the limits of sensitivity for the detection of nucleic acid,there is an obvious advantage to using methods for amplifying therecovered taggant, such as the PCR procedure disclosed in U.S. Pat. Nos.4,683,202 and 4,683,195 and European Patent Publication Nos. 258017 and237362. The PCR method can be used to amplify both single and doublestranded DNA taggants, as well as RNA taggants, and allows for the useof extremely low amounts of taggant, typically of the order 1×10⁻¹¹ to1×10⁻⁶ g per ml of liquid.

PCR amplification can be carried out in a variety of ways, e.g., inverseand asymmetric PCR are well known variations of the technique. Inanother variation, promoters for RNA transcription can be incorporatedinto primers, which, when extended and replicated by PCR, can then beused to create RNA copies of the target sequence. These RNA copies can,in turn, be reverse transcribed into DNA, which can then be amplified byPCR. As with all PCR processes, reaction cycles can be repeated as oftenas desired.

A double stranded taggant is preferred for PCR amplification, although asingle stranded taggant will become double stranded after the firstcycle of amplification, because it is less susceptible to degradation,e.g., by nuclease activity. The taggant preferably has a minimum lengthof about 50 to 70 bases. This permits the hybridization of two primerswhich are typically about each 20 bases in length, and which, whenhybridized to the taggant, are separated by an internal region having alength of from 10 to 30 bases. This internal region is the variableregion responsible for giving each taggant its own unique characteristicsignal. If this region is 10 bases long, then with the four basesavailable for DNA/RNA, approximately 1.048×10⁶ unique taggants can besynthesized. If this region were to be 30 bases in length, approximately1.15×10¹⁸ unique taggants can be synthesized.

An outline taggant is as follows:

1. The taggant DNA could be a synthetic, double stranded DNA sequence of70 to 90 base pairs (bp). ##STR1##

2. The regions AB and CD will be constant for all taggants and willcarry pre-determined sequences which recognise appropriate complementaryprimers for use:

(i) in PCR amplification and,

(ii) in DNA sequencing of PCR amplified DNA.

3. The region BC is the variable region of the microtrace DNAresponsible for its unique, characteristic signal.

4. One or both ends of the taggant may be labelled with biotin to allowthe taggant to be coupled to particles, e.g., microbeads, coated withstreptavidin.

The sequence of a preferred taggant is as follows:

(5') GGC CTA GAA GAA GGT TGA AGC TCC GGG GTA ACG CCA GGG TTT TAC AGT GGTGTT GCC CAA GCC TCC AGC AGC TGT GTA TGC CCA TCT CAT CCA ACC TCT T(3')(SEQ ID NO:1)

Bases 1-25 from 5' end of SEQ ID NO:1 (i.e. GGC CTA GAA GAA GGT TGA AGCTCC G) are from primer G-18 sold by Oligo's Etc. Inc. This is one of theprimers to be used in PCR amplification.

Bases 26-43 (i.e. GG GTA ACG CCA GGG TTT T) from the 5' end of SEQ IDNO:1 are sequencing primer S-27 from Oligo's Etc. Inc. This is thesequence to be used in sequencing the random piece of theoligonucleotide.

Bases 44-75 (i.e. AC AGT GGT GTT GCC CAA GCC TCC AGC AGC TGT) are aslightly modified sequence chosen at random from the STS gene describedin Ballabio et al Nature 393 220 (1990). This is the random sequencewhich gives a unique label with calculable probabilities of only beingthis sequence. Modifications are possible, e.g., position 50 is Ginstead of C; position 56 is C instead of G; and position 75 is Tinstead of G.

Bases 76-100 from the 5' end SEQ ID NO:1 are from the complement of theprimer G-19 sold by Oligo's Etc. Inc. This is the second primer whichenables the complementary strand to be amplified by PCR.

Biotin CPG attached to 3' end, during the synthesis of theoligonucleotide, and this gives the anchoring point for theoligonucleotide to attach to the streptavidin or neutralite coatedparticles.

When detecting nucleic acid by PCR, prior knowledge of the sequence ofthe taggant is necessary to provide appropriate primers. This knowledgeoffers a valuable degree of security for those who desire it, forwithout the primers, which can remain proprietary, the taggant arevirtually undetectable.

For detection of taggants, one can use standard nucleic acidhybridization assays or nucleic acid sequencing. Standard nucleic acidhybridization assays include single phase and mixed phase assays, suchas sandwich assays, and require prior knowledge of the sequence beingdetected to provide the appropriate complementary polynucleotides forcapture or signal purposes.

Alternatively, the nucleic acid recovered from the samples can besequenced using conventional sequencing technology. Commerciallyavailable kits are suitable for this purpose. The basic sequencingtechnology is derived from seminal references, such as the Maxam andGilbert procedure for DNA sequencing described in Methods in Enzymology,Vol. 65, pp. 497 to 559. Sequencing is a more difficult procedure, butoffers greater reliability than nucleic acid hybridization assays. Thisis due to the possibility of contamination by extraneous nucleic acidwith sufficient complementarity to hybridize to the selected probes andoffer false positives.

Based on the outline taggant described above, the following discussionis directed to the particular problems associated with markinghydrophobic liquids, such as oil and other hydrocarbons, using particleshaving, in addition to the aforedescribed signal means (either on thesame or different particles) a unique DNA taggant. The particlespreferably comprise a fluorescent label to enable their detection and/orisolation using a fluorescence microscope or flow cytometer.

Because of the hydrophobic nature of the liquid, the particles should beformed of a material which can be stably dispersed in the liquid,without partitioning into the aqueous phase, and which is inert, i.e.,non-reactive, for that liquid. Especially preferred are beads formedfrom polyacrylates, such as poly(acrylic acid) and poly(methacrylicacid).

The beads preferably have an average diameter no greater than 5 μm, witha typical size of between 0.1 to 1 μm. The density of the beads ispreferably matched with the specific gravity of the oil, in order toprevent sedimentation or precipitation (creaming) and unevendistribution of the label.

DNA can be attached to the chosen hydrophobic beads in a number of ways.Beads such as paramagnetic carboxyl-modified polystyrene beads(Polysciences, Northampton UK) or paramagnetic tosyl-activatedpolystyrene beads (Dynal (U.K.) Ltd.), may also be used in this context.The DNA can be attached covalently by linking the 5' terminal free aminogroup to a suitable target, e.g., the carboxyl group of the carboxylmodified polystyrene. Such techniques are routine (Lund et al., NucleicAcid Research, Vol.16, p. 10861, 1980). Following DNA attachment, thelabelled beads can be washed in water and air dried. The excess carboxylgroups on the beads which have not been bonded to a taggant molecule,can be `capped` with octylamine dissolved in an aqueous solvent, such asdimethylformamide, using dicyclohexylcarbodiimide as the cross-linkingreagent. Alternatively, the taggant may be labelled with biotin and thebeads coated with streptavidin. Excess streptavidin on the beads whichhas not been bonded to a taggant molecule can be capped with freebiotin.

If only a few DNA molecules, but enough for subsequent PCRamplification, sequence analysis and decoding, are added and bonded tothe beads, the proportion of hydrophilic surface (due to the DNA)compared with the overall hydrophobic surface (due to the composition ofthe bead) is normally insufficient to cause the DNA-bead complex topartition into the aqueous phase. The beads remain in the hydrocarbonuntil some procedure is used to remove the bead with its attachedtaggant from the hydrocarbon.

The beads with taggant can be dissolved in solvents, such as chloroform,ether, petroleum ether or toluene, which, in turn, can be dissolved inthe oil to be labelled, ensuring an even distribution of the beads andhence the taggant in the oil. The beads can be separated for evaluationof the label by using magnets to pull the beads into one region fromwhich they can be physically separated, or more simply bycentrifugation.

To ensure that the beads with attached DNA cannot be removed from thehydrocarbon by aqueous washing, the negative charges associated with thephosphodiester structures of the DNA molecule can be removed bymethylation of these groups. Methylation of a region of the DNA moleculewill ensure that this part of the molecule becomes hydrophobic, therebyensuring that the DNA molecule remains within the hydrocarbon phase andis not transferred to the aqueous phase. This can be achieved even ifpart of the DNA molecule retains its negative charge, i.e., isnon-methylated. Methylation of the DNA molecule can be achieved bysynthesis with nucleosides synthesized with methyl phosphonates.

Any procedure which favours solubilisation of DNA molecules inhydrocarbons instead of an aqueous phase could be used as an alternativeto methylation, e.g., by labelling the nucleoside bases of the DNA withbiotin or a hydrophobic hapten, such as fluorescein, dinitrophenol ortri-iodothyronine. Alternatively, sulphonucleotides containingthiophosphates could be incorporated into the taggant and subsequentlyderivatised with thiol-specific modifying agents, such as iodoethanol.

"MPC-1", "MPC-6", "MPC-E", "DYNABEADS", "DYNASPHERES" and "DETACHaBEAD"are all registered trademarks of DYNAL AS of Oslo, Norway.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCCTAGAAGAAGGTTGAAGCTCCGGGGTAACGCCAGGGTTTTACAGTGGTGTTGCCCAA60                GCCTCCAGCAGCTGTGTATGCCCATCTCATCCAACCTCTT100                                   __________________________________________________________________________

We claim:
 1. A method of marking a liquid and subsequently detectingthat the liquid has been marked and identifying the liquid, which methodcomprises:adding to the liquid an additive comprising a plurality ofparticles in an amount no greater than 1 part weight of particles per10⁶ parts weight liquid, said plurality of particles comprising signalmeans to aid their detection, and coding means to aid identification ofthe liquid, said particles not being visible in the liquid to the nakedeye; said additive comprising either (a) two or more particles, eachparticle having a different signal means, and at least one particlehaving a code means or (b) a particle having two or more differentsignal means and at least one code means; one of said signal means beinga non-nucleic acid signal means, and another of said signal means beinga nucleic acid signal means; sampling a portion of the liquid containingsaid additive; detecting the presence in the liquid sample of saidparticles having said non-nucleic acid signal means; detecting thepresence of said nucleic acid signal means on particles from the liquidsample; and decoding said code means, thereby detecting that the liquidhad been marked and identifying the liquid sample.
 2. A method asclaimed in claim 1 in which the particles are present in the liquid inan amount no greater than 1 part weight of particles per 10¹⁰ partsweight liquid and the particles have an average size no greater than 1μm.
 3. A method as claimed in claim 2 in which the particles are presentin :he liquid in an amount of from about 1 part weight of particles per10¹¹ parts weight liquid to about 1 part weight particles per 10¹² partsweight liquid and the particles have an average size of from 0.05 to 1μm.
 4. A method as claimed in claim 3 in which about 10 particles arepresent per ml of liquid.
 5. A method as claimed in claim 1 in which theparticles comprise microbeads or microspheres.
 6. A method as claimed inclaim 1 in which said other signal means is a radiolabel and detectionof the particles comprises the use of a Geiger-Muller tube, ascintillation counter or the fogging of a photographic film.
 7. A methodas claimed in claim 1 in which said other signal means is an enzyme andthe detection of the particles comprises monitoring the reactioncatalysed by the enzyme.
 8. A method as claimed in claim 7 in which theenzyme is selected from the group consisting of acetoacetatedecarboxylase, alcohol dehydrogenase, aldehyde oxidase, alkalinephosphatase or other lyase, amino acid oxidase, arginine decarboxylase,aspartate decarboxylase, ascorbate oxidase, catalase, galactose oxidase,β-galactosidase, glucose oxidase, glutamate decarboxylase, glycollateoxidase, hexose oxidase, horse radish peroxidase, isomerase, lactic aciddehydrogenase, lactate oxidase, luciferase, lysine decarboxylase, malateoxidase, NADH oxidase, oxalate oxidase, pyruvate decarboxylase, pyruvateoxidase, tryptophan oxidase, urate oxidase and xanthine oxidase.
 9. Amethod as claimed in claims 1 in which said other signal means is afluorescent, luminescent, phosphorescent or other label capable ofproducing a photometric signal.
 10. A method as claimed in claim 9 inwhich the fluorescent label is selected from the group consisting ofallophycocyanine, phycocyanine, phycoerythrine, bis-benzamide, coumarin,fluorescein or a derivative thereof, rhodamine or other fluorescent dye,ethidium bromide, and propidium iodide; and detection of the particlescomprises the use of a fluorescence microscope or a flow cytometer. 11.A method as claimed in claim 1 in which the particles are magnetic anddetection of the particles comprises separation and/or concentration ofthe particles using a magnet.
 12. A method as claimed in claim 1 inwhich the additive comprises particles of two or more different coloursor of at least two distinct sizes or shades and the ratio of thedifferently coloured particles or the differently sized or shapedparticles is known.
 13. A method as claimed in claim 1 in which thenucleic acid tag is DNA and the detection of the nucleic acid tagcomprises the use of polymerase amplification, hybridisation and/orsequencing technology.
 14. A method as claimed in claim 1 in which theparticles are formed of a naturally occurring or synthetic polymericresin, a ceramic material or glass.
 15. A method as claimed in claim 14in which the particles are formed from tosyl-activated orcarboxyl-modified polystyrene.
 16. A method as claimed in claim 1 inwhich the particles are coated with a first molecule having a bindingaffinity for a second molecule.
 17. A method as claimed in claim 16 inwhich first molecule is selected from one of the following pairs: anantigen and specific antibody; hormone and hormone receptor; hapten andantihapton; polynucleotide and complementary polynucleotide;polynucleotide and polynucleotide binding protein; biotin and eitheravidin or strepdavidin; enzyme and enzyme cofactor; and lectin andspecific carbohydrate; and the second molecule is the other of saidpair.
 18. A method as claimed in claim 1 in which the liquid is ahydrocarbon, a paint product, an ink, a perfume, a pharmaceutical, afertiliser, a herbicide, a pesticide or an organic solvent.
 19. A methodaccording to claim 1, wherein said plurality of particles comprises (b)a particle having two or more different signal means and at least onecode means.
 20. A method according to claim 1, wherein said plurality ofparticles comprises a type of particle comprising non-nucleic acidsignal means, a sequencing primer, an amplification primer and itscomplementary sequence, a fixed sequence as a nucleic acid signal means,and a variable nucleic acid sequence as a code means for identifyinginformation.
 21. A liquid containing an additive comprising a pluralityof particles added in an amount no greater than 1 part weight particlesper 10⁶ parts weight of liquid, the particles comprising at least twosignal means to aid their detection and not being visible in the liquidto the naked eye, the first signal means comprising a nucleic acid andthe second said signal means being other than a nucleic acid.
 22. Aliquid as claimed in claim 21 in which the additive is an additiveaccording to any of claims 1-5, 7, 8, and 10-18.
 23. A liquid accordingto claim 21 in which said additive is according to claim
 9. 24. A liquidas claimed in claim 21 which is a hydrocarbon, a paint product, an ink,a perfume, a pharmaceutical, a fertiliser, a herbicide, a pesticide oran organic solvent.